No evidence for xenotropic murine leukemia-related virus infection in Sweden using internally controlled multiepitope suspension array serology

Abstract

Many syndromes have a large number of differential diagnoses, a situation which calls for multiplex diagnostic systems. Myalgic encephalomyelitis (ME), also named chronic fatigue syndrome (CFS), is a common disease of unknown etiology. A mouse retrovirus, xenotropic murine leukemia-related virus (XMRV), was found in ME/CFS patients and blood donors, but this was not corroborated. However, the paucity of serological investigations on XMRV in humans prompted us to develop a serological assay which cover many aspects of XMRV antigenicity. It is a novel suspension array method, using a multiplex IgG assay with nine recombinant proteins from the env and gag genes of XMRV and 38 peptides based on known epitopes of vertebrate gammaretroviruses. IgG antibodies were sought in 520 blood donors and 85 ME/CFS patients and in positive- and negative-control sera from animals. We found no differences in seroreactivity between blood donors and ME/CFS patients for any of the antigens. This did not support an association between ME/CFS and XMRV infection. The multiplex serological system had several advantages: (i) biotinylated protein G allowed us to run both human and animal sera, which is essential because of a lack of XMRV-positive humans; (ii) a novel quality control was a pan-peptide positive-control rabbit serum; and (iii) synthetic XMRV Gag peptides with degenerate positions covering most of the variation of murine leukemia-like viruses did not give higher background than nondegenerate analogs. The principle may be used for creation of variant tolerant peptide serologies. Thus, our system allows rational large-scale serological assays with built-in quality control.

Fig 1

Synthetic peptides used in the study. The design of many of the peptides was based on the literature (see references in Materials and Methods).

Fig 2

Overview of internal controls of the serology system. (1) A bead containing thioredoxin A (TrxA) is used to control for anti-TrxA antibodies remaining after absorption with free TrxA. (2) An Escherichia coli lysate-coupled bead controls for anti-E. coli antibodies remaining after absorption with E. coli lysate. Both TrxA- and E. coli-containing beads are necessary for controlling the results with recombinant TrxA fusion proteins prepared in E. coli. (3) Antigen-coated beads are used for surveillance of the antigenicity of individual antigens via rabbit antipeptide hyperimmune serum, of overall antigenic performance via the antigenic index (Fig. 4), and of background binding without serum (nontemplate control [NTC]). (4) The presence of antibodies capable of binding to a common antigen was ensured with the Haemophilus influenzae B (Hib)-containing bead. (5) A “naked” bead, without any bound antigen, was used to control for nonspecific binding to the Luminex beads. Weak nonspecific binding is symbolized with a light gray antibody symbol.

Fig 3

Comparison of the pre- and postimmunization antipeptide sera (dilution, 1:100) received from Genscript. The postimmunization sera reacted more strongly to all peptides than the preimmunization sera. Sera 19 and 22, 20 and 23, and 21 and 24 came from rabbits 1, 2, and 3, respectively. The difference between pre- and postimmunization sera was greatest for rabbit 3, which yielded sera 21 and 24. MFI, median fluorescence intensity.

Fig 4

Successive average antigenicities of 16 indicator peptides with rabbit serum 21, plotted as the percentage of the average median fluorescence intensity in relation to an initial average (of the first six values).

Fig 5

Reactivities (median fluorescence intensity [MFI]) of XMRV-infected and uninfected Mus pahari mice against recombinant p15E protein. Sera were obtained at 2, 5, 8, and 12 weeks postinfection (p.i.). Sera (a kind gift from Yasuhiru Ikeda) were obtained from mice a2, a3, a4, a5, and a6, which were XMRV infected, at 2 and 5 weeks postinfection. All except mouse a4 developed an antibody response. Results with control sera from two other, noninfected, mice (UIC1 and UIC2) are also shown. The top panel shows results with a 1:25 serum dilution, while the bottom panel shows results with a 1:250 serum dilution.

Fig 6

Reactivities (median fluorescence intensity [MFI]) of a serum (diluted 1/25) from XMRV-infected Mus pahari mouse a3 obtained at 5 weeks postinfection. A suspension array of 37 XMRV synthetic peptides and recombinant proteins was used. The ordinate has a log scale. The absence of reactions with the synthetic peptides and presence of reactions with the recombinant p15 (but not the longer version p15L), p10 (but not the longer version p10L), p15E, and gp85 (but not gp70) are illustrated.

Fig 7

Ratio of reactivity (median fluorescence intensity [MFI], shown with a log scale) of a goat anti-p30 hyperimmune serum versus reactivity of a normal goat serum (“ap30_ngoat”) and of a goat anti-gp70 hyperimmune serum versus reactivity of the same normal goat serum (“agp70_ngoat”). Results with beads with synthetic peptides from the capsid protein and recombinant capsid protein (p30) are shown. The sequences of the peptides are shown in Fig. 1. Despite the rather high degeneracy of the two peptides ca0adeg (432 variants) and ca0bdeg (144 variants), they did not give a stronger binding than the nondegenerate counterparts.

Fig 8

Antibody reactivities (median fluorescence intensities [MFI]) to 38 XMRV antigens of sera taken before (pre 24) and after (post 21) immunization of rabbit 3 with an XMRV peptide mixture. Results obtained on three separate runs (1 to 3) on three days are shown. The arrow indicates that in run 3 the gp70 bead did not contain enough of recombinant gp70 protein, an illustration of the utility of this quality control feature. The reactivities of some peptides were rather weak but are evident in the logarithmic presentation. Blue bars are from preimmunization serum 24. Red/magenta bars are from postimmunization serum 21. (A) Linear ordinate scale. (B) Log₁₀ ordinate scale.

Fig 9

Average reactivities (MFI) and standard deviations of 39 XMRV antigens run against 64 ME/CFS sera and 90 blood donor (BD) sera at a dilution of 1/2. Note that even at the low dilution of 1/2, which should favor nonspecific binding, the g1deg (24 variants) and ma0deg (108 variants) degenerate peptides (boxed) did not have significantly higher binding activity than the nondegenerate variants.

Fig 10

Mean reactivities (MFI) of 38 XMRV antigens (synthetic peptides and recombinant proteins). The upper section (ME) shows the reactivities of 85 sera from ME/CFS and fibromyalgia patients, diluted 1/100. The lower section (BD) shows the reactivities of 254 blood donor sera diluted 1/100. The asterisk indicates that two outlying values were removed. The panel of antigens was somewhat different from the ones shown in Fig. 9; this was due to the successive production of new recombinant proteins and limited availability of xMAP beads. The result with the one degenerate peptide in this experiment is boxed.